According to a Return Path analysis of all acquisition campaigns sent through its Postmaster Direct Network over the last two years, click rate goes down dramatically when the subject line is longer than 50 characters (via MediaPost). Click-through rates for emails with subject lines with 49 or fewer characters were 75 percent higher than for those with 50 or more characters.

Open rate differences weren't as dramatic: Subject lines with 49 or fewer characters had open rates 12.5 percent higher than those with 50 or more characters. Return Path points out that testing subject lines is the only way to know what will work and what won't.